Accepted_test

The work describes the study using molecular modeling methods and modification of the recombinant bioluminescent Ca²⁺-regulated ctenophore photoprotein berovin from Beroe abyssicola. Its highest yield is observed when it is incubated with coelenterazine under alkaline conditions and at high salt concentration, while for the hydromedusan photoproteins this occurs under physiological conditions. Ctenophore photoproteins are photosensitive and can be inactivated by irradiation with light of a wide range of wavelengths, whereas this effect is not observed in hydromedusan photoproteins. Berovin is also quite thermolabile and loses its activity at 37°C, which is not typical for jellyfish proteins. Such features of ctenophore photoproteins restrict their applicability as the marker molecules in eukaryotic cell lines. The goal of this work was to predict the amino acid substitutions in berovin that can lead to a shift in activation conditions to the physiological pH values and salt concentration. The probable position of amino acid residues relative to the substrate in the active site was determined by superimposing a model of the spatial structure of berovin obtained using AlphaFold, and modification of the protein was carried out using site-directed mutagenesis. The role of Lys90 and Asn107 in the formation of conditions for the activation of berovin in vitro was experimentally predicted and the mutants with optimal activation under physiological conditions and with increased photostability were obtained.